Project Description:
The rapid surge in drug-resistant bacterial infections has now become one of the primary public health crises of the 21st century. The most problematic infections are caused by drug resistant Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli). Discovery and development of new strategies against the most serious pathogenic bacteria are desperately needed. While traditional drug discovery continues to be an important platform for the search of new antibiotics, alternative approaches should also be pursued to complement these efforts. Towards these aims, our research team will study Gram-negative pathogens by gaining further insight into how bacteria use outer membrane vesicles (OMVs) to promote pathogenesis.
OMVs are nanometer-scale vesicles that are actively released from the outer membrane (OM) of Gram negative bacteria. The OMVs pack a range of biomacromolecules, including toxins, proteins, nucleic acids, and cell wall peptidoglycan (PG) inside host organisms. Over the last few years, it has become increasingly clear that contents packaged within OMVs have direct roles in influencing how bacterial pathogens colonize their hosts and how their hosts respond to bacterial invasion. During bacterial infections, the host innate immune system is under constant surveillance for pathogen associated molecular patterns, including PG. The recent finding that PG is packaged within OMVs has direct consequences on how bacteria may be actively remodeling the host for colonization. Critically, it was further demonstrated that OMV-associated PG activates intracellular receptors to trigger an immune response. Together, these findings point to PG having an important role in how OMVs control pathogenesis. Yet, the organization of PG within vesicles and mechanism by which the fragments of PG control OMV production have not been firmly established.
In the summer of 2017, we made significant strides into further understanding the PG structure within secreted OMVs from Gram-negative pathogens. We used unnatural D-amino acids to tag PGs within OMV with non-native epitopes. Synthetic D-amino acids are chemical probes that enter the PG biosynthetic pathway and are incorporated within live bacterial PG by endogenous enzymes that display broad substrate promiscuities. By tagging bacterial PG within OMVs in live bacterial cells with tailor handles, we have gained unprecedented details about their structure. Our BDSI team was able to show that, contrary to the current hypothesis about OMVassociated PG, bacterial PG is heavily processed within OMVs. Our results showed that PG within OMVs is almost entirely dissembled back to monomeric fragments. Fragmented PG are ligands for several PG sensors in human cells. Therefore, our results suggest that the OMVs are carrying components that may divert the immune response away from the intact cells towards their secreted vesicles. In addition, we made an observation that may yet lead to a new research direction and this observation will be expanded during the summer of 2018. Our research team found that various PG building blocks (PG contain a number of D-amino acids) that are normally secreted into the culture media by bacteria serve to control OMV production levels. If these results are further verified, it may imply that bacterial cells can communicate and coordinate production of OMVs via D-amino acid signaling.
Our team is well positioned to complete the research aims. The Brown research group has experience in the purification and characterization of OMVs produced by various pathogens. The Pires research group has extensive expertise in synthesizing novel D-amino acid building blocks that enter the PG biosynthetic machinery to install unnatural epitopes. The diverse techniques and concepts from each of our laboratories will serve to greatly enrich the experience by the BDSI students. Dr. Brown and Dr. Pires work hard to provide a nurturing and hands-on approach to train new students, giving them the freedom to learn by trial-and-error on their way to becoming truly independent thinkers and researchers. During the summer of 2018, we will pursue two main aims that expand on our previous findings: Specific Aims. To establish the structure and organization of PG within OMVs through the use of PG-specific probes and to demonstrate whether extracellular D-amino acids control OMV production.
We will: a) Confirm our results showing fragmented PG within OMVs across a number of Gram-negative strains b) Determine the actual chemical structure of the PG within OMVs c) Establish whether the PG within OMVs activate human PG-sensors d) Measure OMV production in a series of Gram-negative pathogens when treated with a diverse set of naturally existing D-amino acids By the end of the summer, we aim to have made considerable progress in understanding how PG and PG building blocks (D-amino acids) influence bacterial pathogenesis of Gram-negative bacteria.